Combating the ongoing tuberculosis (TB) epidemic, that claims over 1 million lives per year, is one of the major challenges in global health. Mycobacterium tuberculosis (Mtb) relies on environmental cues to adapt its physiology and promote its survival during infection. Two important host niches colonized by Mtb, the macrophage (M?) and necrotic granuloma, are acidic environments. pH-dependent adaptations are critical to Mtb pathogenesis and may point to new therapeutic targets, but the mechanisms by which pH remodels Mtb physiology remain poorly understood. In preliminary studies we sought to discover selective inhibitors of the pH-inducible two component regulator PhoPR or Mtb survival at acidic pH, which have heretofore been unavailable. Using an innovative, whole-cell phenotypic screen of a >273,000 compound library, we successfully identified chemical probes that inhibit the PhoPR regulon, including the carbonic anhydrase (CA) inhibitor ethoxzolamide (ETZ). Treatment of Mtb with ETZ copies phoPR mutant phenotypes and fully inhibits Mtb CA activity in whole cells, supporting our novel hypothesis that CA physiology modulates PhoPR signaling. We also discovered chemical probes that function independently of PhoPR and selectively inhibit Mtb growth at acidic pH. We hypothesize that many of these probes are targeting physiology that is only essential for growth at acidic pH. Following a series of prioritization assays, we have identified 4 novel chemical probes to use for mechanistic analyses and optimizations. These are innovative chemical genetic tools that will define new pathways required for Mtb survival at acidic pH. Guided by the novel chemical probes that we have discovered, the unifying goal of this application is to define the function and therapeutic potential of chemical probes that modulate pH-driven adaptation via PhoPR-dependent (Aim 1) and PhoPR-independent (Aim 2) mechanisms. These basic and applied studies will define new mechanisms of pH-driven pathogenesis and generate proof-of-concept data supporting the development of new TB therapeutics. The rationale for our proposed studies is that phoPR and pH-driven adaptation are essential for Mtb pathogenesis; hence we predict that studying chemicals and genes impacting these pathways will uncover new pathogenic mechanisms and drug targets. Because we have already discovered novel compounds that function in whole Mtb cells, we are well-positioned to rapidly translate our findings into new drug development candidates. The Specific Aims are to: 1) Define new regulatory mechanisms controlling PhoPR-dependent pathogenesis and determine the suitability of the pathway as a therapeutic target; and, 2) Identify pathways targeted by chemical probes that selectively inhibit Mtb growth at acidic pH, and define their roles in Mtb pathogenesis. Overall Impact: Define new mechanisms of Mtb pH-driven pathogenesis, develop chemical probes targeting these pH-dependent adaptation pathways, and evaluate novel approaches to TB therapy.